Hepatitis B virus (HBV) infects nearly 1/10th of the world population and causes severe liver diseases including cancer. The e antigen (HBeAg) is a secreted soluble protein that promotes immune tolerance during perinatal infection and buffers immunity against HBcAg. The corresponding anti-HBe immune response plays a critical role in the clearance of HBV infection. Therefore, following seroconversion from HBeAg antigenemia to anti-HBe, HBV escape mutants with reduced or no HBeAg production often replace wild-type HBV genomes. The core promoter mutants have various nucleotide changes around positions 1750 -1770 of the HBV genome, and the most common mutations at 1762 and 1764 are known to reduce HBeAg expression at the transcriptional level. The precore mutants have nonsense or frameshift mutations in the HBeAg coding sequence and terminate HBeAg expression at the translational level. Considering the critical importance of HBeAg and anti-HBe in shaping the outcome of HBV infection, we wish to uncover novel regulatory mechanisms for HBeAg expression. In this regard, perinatally infected South African patients seroconvert from HBeAg to anti-HBe at a much accelerated pace than similarly infected Asian patients. Interestingly, the South African HBV variants often harbor two or three point mutations near the precore initiation codon. We plan to verify whether the mutations cause leaky scanning to reduce HbeAg translation. Second, HBeAg maturation requires double proteolytic cleavage events in the secretory pathway. The first cleavage occurs in endoplasmic reticulum to remove the N-terminal signal peptide of 19 residues. A Val to Phe missense mutation at residue 17 is frequently detected in HBV genomes isolated from seroconverted patients. Since an aromatic residue at the -3 position of cleavage site is forbidden, we will test whether this mutation impairs HBeAg cleavage and secretion. Third, we recently identified several naturally occurring core promoter mutants with wild-type level of HBeAg production. Based on the results of preliminary mapping experiments, we will determine whether the number and position of core promoter mutations influence the level of HBeAg expression, and whether missense mutations in the HBeAg coding sequence also regulate HBeAg level. This study promises to verify and identify novel mechanisms regulating HBeAg expression, which has a major impact on the outcome of HBV infection.